程序性死亡受体1和T细胞免疫球蛋白黏蛋白分子3在乳腺癌肿瘤微环境中的表达、分布及其与临床病理特征的关系

Expression and distribution of programmed death receptor 1 and T cell immunoglobulin mucin 3 in breast cancer microenvironment and its relationship with clinicopathological features

目的探讨程序性死亡受体1（PD-1）、T细胞免疫球蛋白黏蛋白分子3（TIM．3）在乳腺癌肿瘤微环境中的表达、分布特征，并分析两者之间的相关性及其与临床病理特征的关系。方法收集嘉兴市第一医院乳腺病科2016年6月至2017年5月收治的30例浸润性乳腺癌确诊患者术后肿瘤组织标本及癌旁组织标本，将标本沿中心分为两份，一份经包埋、冷冻切片后，采用免疫荧光组织染色法观察肿瘤组织中PD-1、TIM-3蛋白表达情况及分布特征，另一部分标本经剪碎、消化后，采用非连续密度梯度离心法提取肿瘤浸润性淋巴细胞（TILs），通过实时荧光定量PCR技术（qRT—PCR）测定TILs中PD-1、TIM-3的mRNA相对表达量，同时通过Western blotting技术测定其蛋白表达量。分析PD-1、TIM-3的表达与乳腺癌临床病理参数的关系，并对PD-1、TIM-3之间进行相关性分析。结果免疫荧光结果可见相对于癌旁组织，肿瘤组织中观察到了较多的PD-1、TIM-3阳性细胞。qRT-PCR显示TILs中PD-1、TIM-3mRNA表达量均显著高于癌旁组织淋巴细胞（3．09±0．38比1．26±0．23、3．42±0．31比1．57±0．29，t=4．16、4．37，均P〈0．05）。蛋白水平上，肿瘤组织淋巴细胞中PD-1、TIM-3的表达水平（0．66±0．08、0．80±0．11）均明显高于癌旁组织（0．10±0．01、0．26±0．02）（t=6．79、4．57，均P〈0．05）。病理资料分析，在肿瘤组织学分级、肿瘤大小、淋巴结转移方面，患者肿瘤组织淋巴细胞PD-1、TIM-3mRNA表达水平差异均有统计学意义（t=2．22-2．99，均P〈0．05）。相关性分析显示，肿瘤组织中PD-1、TIM3的表达之间呈显著正相关（r=0．616，P〈0．01）。结论在乳腺癌肿瘤微环境中，PD-1、TIM-3介导的信号通路对于乳腺癌的发生发展中发挥着重要作用，为乳腺癌的组合靶向治疗提供新的依据。

Objective To explore the expression and distribution of programmed death receptor 1 （PD-1） and T-cell immunoglobulin mucin 3 （TIM-3） in breast cancer microenvironment and analyze the their correlation with the clinicopathological features. Methods The specimens of tumor tissue and adjacent tissues from 30 patients with infiltrative breast cancer who were diagnosed as breast cancer from June 2016 to May 2017 in The First Hospital of Jiaxing were collected, and the specimen were divided into two parts along the center. After embedding and cryosectioning, the expression and distribution of PD-1 and TIM-3 protein in tumor tissues were observed by immunofluorescence staining. Another part of the specimen was cut and digested, and non-continuous density gradient centrifugation was used to extract tumor-infiltrating lymphocytes （TILs）, real-time quantitative PCR （qRT-PCR） was used to detect the mRNA expression of PD-1 and TIM-3 in TILs. Meanwhile, the protein expression was determined by Western blotting. The relationship between the expression of PD-1 and TIM-3 and pathological parameters of breast cancer was analyzed with correlation analysis. Results Immunofluoreseence results showed that more PD-1 and TIM-3 positive cells were observed in the tumor tissues compared with the tumor-adjacent tissues. The qRT-PCR showed that the expression of PD-1 and TIM-3 mRNA in TILs were both significantly higher than those in paracancerous tissues （3.09 ±0. 38 vs 1.26±0. 23, 3.42±0. 31 vs 1.57±0. 29, t = 4. 16, 4. 37, both P 〈 0. 05）. At the protein level, the expression of PD-1 and TIM-3 in tumor tissue lymphocytes （0. 66±0.08, 0. 80±0. 11 ） was significantly higher than those in cancerous tissues （0. 10±0. 01, 0. 26±0. 02） （t = 6. 79, 4. 57, both P 〈 0.05 ）. There were significant differences in the expression of PD-1, TIM-3 mRNA in the TILs between the different tumor histological grades, tumor sizes, lymph node metastasis （t = 2. 22-2. 99, all P 〈 0.05 ）. Correlation analysis showed that there was a significant positive correlation between the expression of PD-1 and TIM-3 in tumor tissues （r = 0. 616, P 〈 0. 01 ）. Conclusions In the breast cancer microenvironment, PD-1, TIM-3-mediated signaling pathway plays an important role in the occurrence and development of breast cancer, it provides a new basis for the combination therapy of breast cancer.